1.1 Field of the Invention
The present invention relates generally to the fields of cardiovascular disease and hypertension. More particularly, it concerns antisense oligonucleotide compounds that specifically bind to mRNA encoding a mammalian angiotensin converting enzyme (ACE) and inhibit its expression. Disclosed are antisense oligonucleotide and peptide nucleic acid compositions, pharmaceutical formulations thereof, and vectors encoding antisense oligonucleotides that specifically bind ACE mRNA and alter ACE expression in a host cell. Also disclosed are methods for making and using these antisense molecules, compositions, and vectors to reduce ACE activity, decrease translation of ACE-specific mRNA, and treat disorders in a mammal arising from elevated ACE expression, including hypertension.
1.2. Description of Related Art
1.2.1 Hypertension
Hypertension is the result of increased arterial resistance to blood flow and left untreated can lead to various pathological consequences. Hypertension affects approximately 40 million people in the United States. Heart attack (Nicholls et al., 1998), kidney damage (Agodoa, 1998), stroke (Chamorro et al., 1998) and loss of vision (Satllworth and Waldron, 1997) are typical conditions that result from high blood pressure. When blood vessels are subjected to high pressure for extended periods of time, they respond by thickening, vasospasm, and internal build-up of lipids and plaques, a condition known as arteriosclerosis. Arteriosclerosis further causes a decreased blood flow to the kidneys, which respond by releasing the protease renin. An overactive renin-angiotensin system is often implicated in the development of hypertension and cardiovascular disease (Nicholls et al., 1998).
Hypertension is often called the "silent killer," since half of the population afflicted with high blood pressure are unaware of the condition. Thus, an initial step in combating hypertension is early detection. Following diagnosis, actions must be taken to control the disorder. Diuretics, beta-adrenergic blockers, calcium channel blockers and ACE inhibitors are all commonly administered drugs used to treat hypertension. Each of these drugs has negative side effects associated with it, limiting their use in many situations. For example, the commonly prescribed ACE inhibitors captopril, enalapril and lisinopril have demonstrated a variety of adverse side effects such as cough, rash, fetal death, intra-uterine growth retardation, hypotension, acute renal failure and hepatotoxicity (Sadeck et al., 1997; Deira et al., 1997; Menefee et al., 1998). Further, the short lasting effect of these drugs requires a daily dose. Poor compliance is a major problem with drug regimens and can lead to a hypertensive crisis if the drug is not taken as scheduled.
1.2.2 The Renin-Angiotensin System (RAS)
Angiotensinogen (AGT), a protein produced mainly in the liver, is the substrate for the enzyme renin, produced by the kidneys. The action of renin on AGT is the formation of the decapeptide angiotensin I. Angiotensin I is hydrolyzed by angiotensin converting enzyme (ACE), yielding the octapeptide angiotensin II (Ang II). Ang II is considered to be one of the major contributors to hypertension (Alderman et al., 1991) and is also a growth stimulator of heart tissue and vascular smooth muscle cells (Dzau, 1993). Ang II is not only a potent vasoconstrictor and regulator of blood pressure, it may also result in cardiac hypertrophy and possibly play a role in arterial restenosis after angioplasty (Powell et al., 1989). Thus, through the inhibition of ACE, the production of Ang II can be effectively reduced, resulting in a decrease in hypertensive blood pressures and possibly other cardiovascular diseases attributed to excess Ang II production.
1.2.3 Treatments of Hypertension
Currently, four major categories of hypertensive drugs are administered to treat high blood pressure: (1) Diuretics, typically the drug of choice when the abnormal blood pressure is not very high, increase the rate at which the body eliminates urine and salt, resulting in decreased blood pressure by reducing volume (Moser, 1998); (2) .beta.-adrenergic blockers, typically prescribed in combination with diuretics, lower blood pressure and heart rate (Rodgers, 1998); (3) Calcium channel blockers work by preventing the entry of calcium into cells, which reduces vasoconstriction (Rosenthal, 1993); (4) ACE inhibitors prevent the narrowing and constriction of blood vessels by blocking the production of the vasoconstrictive peptide angiotensin II, a product of ACE (Rosenthal, 1993).
1.2.4 Antisense Oligonucleotides
Antisense oligodeoxynucleotides (AS-ODNs) are single-stranded, short sequences of DNA (Cohen, 1989; De Mesmaeker et al., 1995) that are complementary to specific messenger RNA (mRNA). Since AS-ODNs hybridize with the mRNA, they prevent the targeted mRNA from expressing its polypeptide product in the cell.
AS-ODNs have previously been used for the treatment of hypertension (Phillips, 1997; Wielbo et al., 1994; Phillips et al., 1994 and Gyurko et al., 1997), using AS-ODNs targeted to AT-1 angiotensin receptor mRNA (Gyurko et al., 1993 and Meng et al., 1994) and angiotensinogen mRNA protein (Wielbo et al., 1997; Wielbo et al., 1996; Wielbo et al., 1995), that inhibit the synthesis of the AT-1 angiotensin receptor and angiotensinogen protein, respectively. The study by Phillips et al. (1994) confirmed that inhibition of the brain renin-angiotensin system in spontaneously hypertensive rats (SHR) lowers blood pressure, but this work did not extend to the other enzymes in the RAS, such as ACE.
1.3 Deficiencies in the Prior Art
Currently, the art does not provide pharmacological approaches to treating hypertension and abnormalities associated with ACE activity in a cell that avoids these and other problems associated with traditional ACE-inhibitor therapies. Thus, the need exists for an effective hypertensive treatment that circumvents the toxic side effects and provides more specific ACE inhibition with longer acting effects to improve patient compliance. In addition, methods for delivery of antisense oligonucleotides to a host cell, and in particular, non-invasive administration of ACE-specific antisense constructs to a mammal are particularly desirable.